High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

Podsumowanie

Yeast growth phenotypes are precisely measured through highly parallel time-lapse imaging of immobilized cells growing into microcolonies. Simultaneously, stress tolerance, protein expression, and protein localization can be monitored, generating integrated datasets to study how environmental and genetic differences, as well as gene-expression heterogeneity among isogenic cells, modulate growth.

Streszczenie

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental inputs into stress tolerance, pathogenicity, and other key components of fitness. This manuscript describes a microscope-based assay that tracks approximately 10⁵ Saccharomyces cerevisiae microcolonies per experiment. After automated time-lapse imaging of yeast immobilized in a multiwell plate, microcolony growth rates are easily analyzed with custom image-analysis software. For each microcolony, expression and localization of fluorescent proteins and survival of acute stress can also be monitored. This assay allows precise estimation of strains' average growth rates, as well as comprehensive measurement of heterogeneity in growth, gene expression, and stress tolerance within clonal populations.

Wprowadzenie

Growth phenotypes contribute critically to yeast fitness. Natural selection can efficiently distinguish between lineages with growth rates differing by the inverse of the effective population size, which can exceed 10⁸ individuals¹. Furthermore, variability of growth rates among individuals within a population is an evolutionarily relevant parameter, as it can serve as the basis for survival strategies such as bet hedging^2,3,4,5,6. Therefore, assays that allow for highly a....

Protokół

1. Preparation of Randomized Plates (Prior to Experiment Day)

1. Plan the strains and conditions to be tested with the growth assay. At this point, randomly assign strains and conditions to any well.
   NOTE: When considering plate setup, it is advisable to include more than one replicate per strain and growth condition on a single plate to account for well-related noise in measurements. See Discussion for more details.
2. Computationally randomize the location of each strain and environmental condition for plate replicates that will be run on different days.
3. Grow all cells that will be used in the experiment to saturation in yeast extrac....

Wyniki

The novelty of this protocol is that growth rate can be calculated for individual cells within a population by tracking their growth into microcolonies through time-lapse imaging (Figure 2A). Because microcolonies grow for many hours in a planar manner due to the presence of concanavalin A, their areas can be tracked throughout the experiment, and a linear fit to the change in the natural log of the area over time can be used to calculate growth rate for each individual colony observed

Dyskusje

The protocol described here is a versatile assay that allows cell growth and gene expression to be monitored simultaneously at the level of individual microcolonies. Combining these two modalities yields unique biological insights. For example, previous work has used this assay to show a negative correlation between expression of the TSL1 gene and microcolony growth rate in isogenic wildtype cells by measuring both simultaneously^7,10. It is also possible.......

Materiały

Name	Company	Catalog Number	Comments
General Materials
500 mL Bottletop Filter .22 µm PES Sterilizing, Low Protein Binding, w/45mm Neck	Fisher	CLS431154	used to filter the media
BD Falcon*Tissue Culture Plates, microtest u-bottom	Fisher	08-772-54	96-well culture tubes used to freeze cells, pre-grow cells, and dilutions
BD Syringes without Needle, 50 mL	Fisher	13-689-8	Used to filter the Concanavalin A
Costar Sterile Disposable Reagent Reservoirs	Fisher	07-200-127	reagent reservoirs used to pipette solutions with multichannel pipette
Costar Thermowell Aluminum Sealing Tape	Fisher	07-200-684	96-well plate seal for pre-growth and freezing
lint and static free Kimwipes	Fisher	06-666A	lint and static free wipes to keep microscope plate bottom free of debris and scratches
Nalgene Syringe Filters	ThermoFisher Scientific	199-2020	0.2 μm pore size, 25 mm diameter; used to filter concanavalin A solution
Media Components
Minimal chemically defined media (MD; 2% glucose)			alternative microscopy media used for yeast pre-growth and growth during microscopy
Synthetic Complete Media (SC; 2% glucose)			microscopy media used for yeast pre-growth and growth during microscopy
Yeast extract-peptone-dextrose (YEPD; 2% glucose) medium			cell growth prior to freezing down randomized plates
Microscopy Materials
Breathe-Easy sealing membrane	Millipore Sigma	Z380059-1PAK	breathable membranes used to seal plate during microscopy experiment. At this stage breathable membranes are reccomended because they prevent condensation in the wells and allow for better microscopy images
Brooks 96-well flat clear glass bottom microscope plate	Dot Scientific	MGB096-1-2-LG-L	microscope plate
Concanavalin A from canavalia ensiformis (Jack Bean), lyophilized powder	Millipore Sigma	45-C2010-1G	Make 5x concanavalin A solution and freeze 5ml of 5x concanavalin A in 50 mL conical tubes at -80 °C
Strains Used
MAH.5, MAH.96, MAH.52, MAH.66, MAH.11, MAH.58, MAH.135, MAH.15, MAH.44, MAH.132			Haploid mutation accumulation strains in a laboratory background, described in Hall and Joseph 2010
EP026.2A-2C			Progeny of the ancestral Hall and Joseph 2010 mutation accumulation strain, transformed with YFR054cΔ::Scw11P::GFP
Equipment
Misonix Sonicator S-4000 with 96-pin attachment			Sonicator https://www.labx.com/item/misonix-inc-s-4000-sonicator/4771281
Nikon Eclipse Ti-E with Perfect Focus System			Inverted microscope with automated stage and autofocus system